Method and apparatus for checking automated optical system performance repeatability

ABSTRACT

A test checks for appropriate positioning of priority fields for image collection and evaluation. The tests characterize lateral repeatability of stage movement in an X-Y plane, the longitudinal repeatability of the stage along a Z axis, cross coupling of motion in the Z direction from the X-Y stage movement, repeatability of the microscope objective turret positioning, mechanical centration of optical paths and the parfocality of optical paths. The process includes moving to a rough location, performing focus pans to determine the best focus and searching for a known object to register coordinate locations, processing those locations to determine the repeatability and accuracy of the motion system. Further, a means of evaluating these parameters is disclosed by which an automated cytology instrument will validate or invalidate data taken since the last position integrity check.

FIELD OF THE INVENTION

The present invention relates to an automated method for evaluation ofpositioning system performance in automated machine vision instruments.More specifically, the invention provides an automated test methodconducted during operation of an automated microscope system. Still morespecifically, the automated test of the invention characterizes lateralrepeatability of stage movement in an X-Y plane, longitudinalrepeatability of the stage along a Z axis, cross coupling of motion inthe Z direction from movement in the X-Y plane, repeatability ofmovement of a microscope objective turret, mechanical centration ofoptical paths, and parfocality of optical paths in an instrumentperforming automated analysis of biological specimens such as, forexample, Pap smears.

BACKGROUND OF INVENTION

Automated analysis of biological specimens requires a high degree ofrepeatability and accuracy from the motion systems that positionspecimens in the instrument. Repeatability and accuracy errors candecrease throughput and, in the worst case, cause low prevalence data tobe missed. Therefore, it is critical that motion systems employed inautomated biological analysis machines perform above or beyond theengineered limits of the design.

For automated biological analysis applications, such as for Pap smearanalysis, repeatability of movement of a microscope slide stage in theX,Y plane, or horizontal plane, is extremely important. In such systemsprioritized images may be selected under low power magnification and arerelocated under high power magnification for review. In one example ofan automated biological analysis system as manufactured by NeoPath, Inc.of Bellevue, Wash. a low power 4× field of view is divided into a 5×5matrix of high power 20× fields. Each 4× subfield (or 20× field) isanalyzed for further review. If the results dictate further inspection,the system reviews the subfield with the 20× magnification. Thus, stagerepeatability becomes most critical when an object of interest in a 4×subfield lies near the subfield boundary. In such a case, poor XY stagerepeatability may cause the high power 20× review to miss a suspectobject. Therefore, it is one motive of the present invention to providean X,Y repeatability test. As contemplated by the present invention, anX,Y repeatability test is conducted to verify that stage performancemeets engineered limits.

The method of the present invention ensures that priority fields of thelow power scan are appropriately positioned under a high power objectivefor image collection and evaluation. The invention provides a processand apparatus suitable for characterizing lateral repeatability of theX-Y stage, the longitudinal repeatability of the Z stage, the crosscoupling of motion in the Z direction from the X-Y stage, therepeatability of the microscope objective turret, the mechanicalcentration of optical paths and the parfocality of optical paths. Thisprocess involves moving to a rough location, performing focus pans todetermine the best focus and searching for a known object to registercoordinate locations, processing those locations to determine therepeatability and accuracy of the motion system. Further, a means ofevaluating these parameters is disclosed by which the automated cytologyinstrument will validate or invalidate data taken since the lastposition integrity check.

In a presently preferred embodiment of the invention, the camera systemdisclosed herein is used in a system for analyzing cervical pap smears,such as that shown and disclosed in U.S. patent application Ser. No.07/838,064, entitled "Method For Identifying Normal BiomedicalSpecimens", by Alan C. Nelson, et al., filed Feb. 18, 1992; U.S. patentapplication Ser. No. 07/838,395, entitled "Method For IdentifyingObjects Using Data Processing Techniques", by S. James Lee, et al.,filed Feb. 18, 1992; U.S. patent application Ser. No. 07/838,070, nowU.S. Pat. No. 5,315,700, entitled "Method And Apparatus For RapidlyProcessing Data Sequences", by Richard S. Johnston, et al., filed Feb.18, 1992; U.S. Patent Application Ser. No. 07/838,065, filed February18, 1992, entitled "Method and Apparatus for Dynamic Correction ofMicroscopic Image Signals" by Jon W. Hayenga, et al.; and U.S. patentapplication attorney's docket No. 9/1799, filed Sep. 7, 1994 entitled"Method and Apparatus for Rapid Capture of Focused Microscopic Images"to Hayenga, et al., which is a continuation-in-part of application Ser.No. 07/838,063 filed on Feb. 18, 1992 the disclosures of which areincorporated herein, in their entirety, by the foregoing referencesthereto.

SUMMARY OF THE INVENTION

The present invention provides a test that checks for appropriatepositioning of priority fields selected by a first low power scan andrescanned under a high power objective for image collection andevaluation. The invention provides a process and apparatus suitable forcharacterizing lateral repeatability of motion of a stage in an X-Yplane, the longitudinal repeatability of the stage motion along a Zdirection, cross coupling of motion in the Z direction from the stagemovement in the X-Y plane, repeatability of microscope objective turretmotion, mechanical centration of optical paths and the parfocality ofoptical paths.

In one aspect of the invention, the process includes the steps of movingto a rough location, performing focus pans to determine the best focusand searching for a known object to register coordinate locations,processing those locations to determine the repeatability and accuracyof the motion system. Further, a means of evaluating these parameters isdisclosed by which an automated cytology instrument or the like willvalidate or invalidate data taken since the last position integritycheck.

It is one object of the invention to provide a means to characterize XYstage repeatability.

It is another object of the invention to provide a means to characterizeXY stage repeatability during run time with no additionalinstrumentation.

It is yet a further object of the invention to provide a means tocharacterize Z stage cross coupling.

It is still a further object of this invention to provide a means tocharacterize Z stage repeatability.

It is yet another object of this invention to provide a means tocharacterize turret repeatability.

It is still a further object of this invention to provide a means tocharacterize centration and parfocality.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art through the description ofthe preferred embodiment, claims and drawings herein wherein likenumerals refer to like elements.

BRIEF DESCRIPTION OF THE DRAWINGS

To illustrate this invention, a preferred embodiment will be describedherein with reference to the accompanying drawings.

FIG. 1A and FIG. 1B show an automated cytology system and the placementof a calibration and test target into an optical path of an automatedmicroscope as employed by the method and apparatus of the invention.

FIG. 2 schematically shows an automated microscope of the type used inautomated cytological system having a calibration plate mounted on amovable stage.

FIG. 3 shows one example of a calibration and test target or plate asused in one aspect of the invention.

FIG. 4 shows an example of a fiducial marking.

FIG. 5 shows an example of a scan pattern of a point on the XY stageused by one method of the invention.

FIGS. 6A and 6B show a flow diagram of one method of the invention forchecking stage movement repeatability.

FIGS. 7A and 7B show a flow diagram of one method of the invention forchecking repeatability of movement along a Z axis.

FIG. 8 showing a flow diagram of one method of the invention forchecking repeatability of turret movement.

FIG. 9 shows a flow diagram of one method of the invention for checkingobjective centration and parfocality.

FIG. 10 is a graph illustrating the relationship between the passbandfrequency component of the signal provided by the camera assembly ofFIG. 1A and the focus of the camera assembly.

FIG. 11 is a more detailed illustrative diagram of the camera assemblythat comprises the subject invention.

FIG. 12 is an illustrative diagram of a circuit for determining thefocus position of the camera assembly of FIG. 11.

FIG. 13 shows a schematic view of a typical cell.

FIG. 14 shows a process for converting physical cell size intoelectrical band width.

FIG. 15 graphically illustrates a time vary voltage of a dark nucleus.

FIG. 16 shows an inverted pulse representing a square function.

FIGS. 17 and 18 show a Fourier transformation for a square function asemployed in one aspect of the invention.

FIG. 19 illustrates a filter response sensitive to objects of interest,such as cell nuclei as employed in one embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention consists of a suite of tests to characterize theperformance of the motion system during operation without the use of anyadditional instrumentation. These tests refer specifically to an openloop stepper motor lead screw driven system. However, the concepts canbe employed to other motion systems using components such as DC motorsservo motors, linear motors, belt driven systems, and other similarmotive devices.

Now refer to FIGS. 1A and 1B which show a schematic diagram of oneembodiment of the apparatus of the invention for checking positionintegrity for an automated microscope. While the method and apparatus ofthe invention will be discussed in terms of an example herein related toan automated cytology apparatus, it will be understood that theinvention is not so limited. The features and principles of theinvention may be applied to check urine analysis processes,semiconductor process defects, liquid crystal devices and other types ofprocessing systems employing, for example, continuous arc lamps,filament lamps, laser sources, tube cameras, PIN diodes andphotomultiplier tubes.

The apparatus of the invention comprises an imaging system 502, a motioncontrol system 504, an image processing system 536, a central processingsystem 540, and a workstation 542. The imaging system 502 is comprisedof an illuminator 508, imaging optics 510, a CCD camera 512, anillumination sensor 514 and an image capture and focus system 516. Theimage capture and focus system 516 provides video timing data to the CCDcameras 512, the CCD cameras 512 provide images comprising scan lines tothe image capture and focus system 516. An illumination sensor intensityis provided to the image capture and focus system 516 where anillumination sensor 514 receives the sample of the image from the optics510. In one embodiment of the invention, the optics may further comprisean automated microscope. The illuminator 508 provides illumination of aslide. The image capture and focus system 516 provides data to a VME bus538. The VME bus distributes the data to an image processing system 536.The image processing system 536 is comprised of field-of-view processors568. The images are sent along the image bus 564 from the image captureand focus system 516. A central processor 540 controls the operation ofthe invention through the VME bus 538. In one embodiment the centralprocessor 562 comprises a Motorola 68030 CPU. The motion controller 504is comprised of a tray handler 518, a microscope stage controller 520, amicroscope turret controller 522, and a calibration slide 524. The motordrivers 526 position the slide under the optics. A bar code reader 528reads a barcode located on the slide 524. A touch sensor 530 determineswhether a slide is under the microscope objectives, and a door interlock532 prevents operation in case the doors are open. Motion controller 534controls the motor drivers 526 in response to the central processor 540.An Ethernet (™) communication system 560 communicates to a workstation542 to provide control of the system. A hard disk 544 is controlled byworkstation processor 550. In one embodiment, workstation 542 maycomprise a Sun Sparc Classic (™) workstation. A tape drive 546 isconnected to the workstation processor 550 as well as a modem 548, amonitor 552, a keyboard 554, and a mouse pointing device 556. A printer558 is connected to the Ethernet (™) network 560.

During position integrity checking, the central computer 540, running areal time operating system, controls the automated microscope and theprocessor to acquire and digitize images from the microscope. Theflatness of the slide may be checked, for example, by contacting thefour corners of the slide using a computer controlled touch sensor. Thecomputer 540 also controls the microscope stage to position the specimenunder the microscope objective, and from one to 15 field of view (FOV)processors 568 which receive images under control of the computer 540.

Referring now to FIG. 2, there shown is placement of a calibration andtest target 1 into an optical path of an automated microscope 3 having aturret 22. The calibration and test target may be mounted on a stage 521substantially in a horizontal X,Y plane which intersects the opticalpath. The stage 521 is movable in the X,Y plane as well as along a Zaxis which is perpendicular to the X,Y plane and which is parallel tothe optical axis of the automated microscope. The turret 22 may comprisemultiple objective lenses as is well known in the art. The microscopeturret control 522 provides signals in a well known manner forpositioning a selected objective lens into position for viewing a slide,for example.

Referring now to FIG. 3 one example of a calibration and test target isshown. Several of the processes employed by the present inventionrequire a calibration and target plate. In the case of a transmissionmicroscope, the calibration and test target 1 may comprise a piece ofglass approximately 1.45 mm thick. The calibration and test targetadvantageously comprises specified clear areas 34 and image primitivessuch as horizontal and vertical bar targets 36. Other types ofcalibration markings, such as fiducial markings, may also be used. FIG.4 shows an example of a fiducial marking. Such calibration and testtarget plates may be used for most transmission microscopes to simulatethe optical path difference effects introduced by the substrate,coverslip and specimen media. In some embodiments of the invention, thecalibration and test target may be advantageously mounted onto aconventional cantilever arm for ease of placement onto and removal fromthe stage.

X,Y Repeatability and Z Cross Coupling Test

Referring now to FIGS. 6A and 6B, a flow diagram of one method of theinvention for checking stage movement repeatability is shown. At step 62an objective lens of a first magnification power is selected. In oneexample, a 20×magnification is selected. At step 64 the calibrationplate is inserted into the optical path as shown, for example, in FIG.2, and the 0,0 fiducial, best shown in FIG. 3 is located and centered inthe microscope's field of view at step 66. Generally, step 66 maycomprise a number of process substeps for grossly positioning the stagein the vicinity of the 0,0 fiducial as shown at substep 70, performing afocus pan at substep 72, determining the location of the center of thefiducial with respect to the optical axis (or camera center) at substep74 and translating the stage in the X and Y plane to center the fiducialin the field of view at substep 76.

In one example of the invention, a focus pan 72 may comprise the stepsof moving the stage in the Z direction to an estimated focus positionfollowed by incrementally moving towards a position of best focus whileacquiring images at each position. The images acquired during the focuspan are processed for focus features, such as high frequency content.The stage continues to move incrementally until the position of bestfocus has been passed, that is the focus features cease to improve. Thestage is then returned to the position of best focus. Other methods areknown by those skilled in the art to perform a focus pan.

At step 68, the location of the fiducial center is recorded. At step 78the stage is moved in a star like pattern first in a direction away fromthe center of the field of view, then in a direction returning thefiducial to the center of the field of view.

In one example, the stage is automatically moved so as to move thefiducial away from and then back to the center of the field of view on a1.5 mm radius in 15 degree increments for twenty four repetitions. Themotion profile appears to be a star like pattern as shown in FIG. 5.

The basic premise of the method of the invention is to move the fiducialso as to approach a predetermined position from multiple directions todetermine the multidirectional repeatability of the system. The processcontinues with step 80 where, each time the stage returns to the centralposition, the fiducial image is captured by the camera. At step 80 thefiducial image is captured by the camera and the coordinates of thefiducial image are recorded. The recorded coordinate information is usedto determine the lateral position in the X and Y plane of the center ofthe fiducial with respect to the optic axis at step 82. In addition, atstep 84, the Z position of the image is also determined using the focusapparatus described in a co-pending patent application entitled METHODAND APPARATUS FOR RAPID CAPTURE OF FOCUSED MICROSCOPIC IMAGES asdescribed below with reference to FIGS. 10-19. A difference in Zposition from iteration to iteration represents a cross coupling ofmotion into the Z axis from an X-Y move. At step 84 a data array isgenerated comprising the X, Y and Z coordinate for each iteration of thetest. The array is read to determine the maximum and minimum coordinatein each of the X, Y and Z axes for each of the incremental movementsfollowing the star like pattern. The absolute value of the differencebetween each of the corresponding maximum and minimum coordinates isused to determine the repeatability of stage movement in each axis.

The method of the invention is superior to linear encoders orinterferometric methods because repeatability is determined exactly atthe point of interest. Other methods infer position based on a devicethat is remotely located from the point of interest. In addition themethod of the invention allows a quick check of stage performancebetween processing operations performed by the machine being tested withno special instrumentation needed. Table 1 shows the output of the testincluding the limits for test parameters.

                  TABLE 1                                                         ______________________________________                                        X/Y Repeatability and Z Cross Coupling Test                                   Parameter    Result        Limit                                              ______________________________________                                        X repeatability:                                                                           3299          <15000   nm                                        Y repeatability:                                                                           2200          <15000   nm                                        Z cross coupling                                                                           111           <2000    nm                                        ______________________________________                                    

Z Repeatability Test

Refer now to FIGS. 7A and 7B where a flow diagram of one method of theinvention for checking repeatability of movement along a Z axis isshown. Repeatability in Z influences the speed of processing of anautomated microscopy instrument. This is due to the manner in which theinstrument collects images for processing. When an image does not havesuitable focus for processing, a projection of best focus is made andthe image is returned to the queue to be collected later. When thesystem returns to that image the focus projection is applied. If thestage has poor repeatability of movement along the Z axis relative tothe focus error budget, the system may move to the incorrect position,thereby requiring numerous iterations developing a new focus projection,replacing the image back into the queue and reattempting to correctlyposition the image to finally attain acceptable focus. This increasesthe time to process the slide. The Z repeatability test as run inaccordance with the present invention characterizes this error.

Process steps 92, 94 and 96 set up the test and are similar to those asabove described above with reference to FIG. 6A with respect to processsteps 62,64 and 66 respectively. As above, in one example an objectivelens having a 20× magnification is selected and a fiducial 0,0 isfocused and centered in the field of view to establish an origin. Atstep 98 the stage is moved in a negative direction, where a negativedirection is considered to be a direction away from the microscopeobjective. In one useful example, the stage is moved along the Z axis afirst distance of about 10 microns. At step 100 the stage is returned tothe origin. After returning, the actual Z position is analyzed at step100 using the autofocus method and apparatus as described herein. Atstep 104 the stage moved in a negative direction equal to a seconddistance. In the example described herein the second distance may beabout 100 microns. As before, at step 106 the stage returns to theorigin. The image is again processed for Z position at step 108.

The process is repeated again in the positive Z direction beginning atstep 112 where the stage is moved along the Z axis in a positivedirection (that is, toward the microscope objective) a third distance.At step 114 the stage is returned to origin and the actual position ofthe fiducial along the Z axis is measured at step 116. At step 118 thestage is moved in a positive direction for a fourth distance. At step120 the stage is returned to the origin. After returning, the actual Zposition is again analyzed at step 122 using the autofocus method andapparatus as described herein.

Then the process is repeated again in both directions, each time the Zcoordinate is stored at step 124. The multiple distance moves areincorporated to ensure the repeatability in move length independent.After all iterations have been completed, the data array is processed todetermine the difference between the minimum and maximum Z coordinate.An absolute value of the difference is taken as the repeatability of theZ stage. Some results of this test have been generated for the NeoPathsystem and are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Z Repeatability                                                               Parameter        Result  Limit                                                ______________________________________                                        Z repeatability: 149     <2000 nm                                             ______________________________________                                    

Turret Repeatability Test

Refer now to FIG. 8 showing a flow diagram of one method of theinvention for checking repeatability of turret movement. In a similarfashion to the checks for stage repeatability in the X,Y plane, turretmovement repeatability may also affect the efficacy of an automatedmicroscope based instrument. Therefore, the invention provides a turretrepeatability test. The system is set up as before. In this casehowever, the XY and Z stage remains stationary and the turret 22 ismoved out of and back into position. The turret positioning movement isrepeated six times, each time alternating between clockwise and counterclockwise directions. As described above, at step 130 an objective lensof a selected magnification characteristic is used. At step 132 acalibration plate is inserted into the optical path and, at step 134,each time the turret is moved back into position, an image of fiducial0,0 is acquired. The image is processed at step 138 to determine thecenter in the X, Y plane relative to the optical axis of the microscope.In one example, a 6×2 array is developed of X and Y coordinates for eachof the six iterations. The maximum and minimum extent are determined foreach axis. An absolute value of the largest difference is taken as theturret repeatability. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Turret Repeatability                                                          Parameter        Result  Limit                                                ______________________________________                                        Turret repeatability:                                                                          1650    <7000 nm                                             ______________________________________                                    

Objective Centration and Parfocality Test

Refer now to FIG. 9 where a flow diagram of one method of the inventionfor checking objective centration and parfocality is shown. Multipleobjectives are typically used in an automated microscope basedinstrument to vary the magnification. Each objective has its own opticalaxis, that is to say each objective looks at a certain area of thespecimen. It is necessary to make these optic axes collinear such thatthe center of the image in one objective is very close to the center ofanother objective when it is placed in position. In addition, it isadvantageous to make the focal planes coplanar. A test is performed toensure that the optic axes of each objective are collinear and the focalplanes are coplanar.

Process steps 142, 144 and 146 set up the test and are similar to thoseas above described above with reference to FIG. 6A with respect toprocess steps 62, 64 and 66 respectively. As above, in one example anobjective lens having a 20× magnification is selected and a fiducial 0,0is focused and centered in the field of view to establish an origin. Atstep 148, a second objective is selected having a second magnificationcharacteristic. In one example, the system is reconfigured to the 4×magnification and the center of the fiducial is found and focused. Atstep 152, the 0,0 fiducial is again located and centered. The X, Y and Zcoordinates are compared to those of the first selected objective. The Xand Y difference is taken as the centration of the objectives and the Zdifference is taken as the parfocality at step 154. The results arecompared against limits as seen in Table 4.

                  TABLE 4                                                         ______________________________________                                        Centration and Parfocality                                                    Parameter         Result  Limit                                               ______________________________________                                        Centration 4X to 20X                                                                            4400    <25000 nm                                           Parfocality 4X to 20X                                                                           750     <15000 nm                                           ______________________________________                                    

In order to promote further understanding of the invention, one exampleof a method employed by the invention for focussing will now be furtherdescribed. As described above with reference to FIGS. 1A, 1B and FIG. 2,the motion controller 504 includes a stage for receiving the slide 1 andis responsive to a slide scan signal, received from a processor 540, formoving the stage in a slide plane represented by X and Y directions. Inthe illustrative diagram of FIGS. 1A, 1B and FIG. 2, the X and Ydirections are located in the plane that is perpendicular to an opticalpath intermediate the slide 1 and the condenser 402. The motioncontroller 504 is further responsive to a slide focus signal for movingthe slide 1 in a direction normal to the slide plane, along the opticalpath 110, for focusing the camera upon the slide 1. The motioncontroller 504 is constructed to provide a position signal to theprocessor 540 wherein the position signal is indicative of the X, Y, andZ position of the slide 1. Motion controllers for performing theabove-described functions are known in the art and a suitable motioncontroller may be selected by those skilled in the art.

The camera assembly 512 is constructed to provide an image signalindicative of the optical transmissivity of the specimen on the slide 1.The image signal from the camera assembly 512 is obtained by focusingthe camera assembly 512 on a focal point positioned a first distancealong the optical path 110. The camera assembly 512 is furtherconstructed to provide an above focus image signal and a below focusimage signal, referred to herein as a focus plus and a focus minussignal, respectively. The focus plus signal is provided by focusing thecamera assembly on a focal point positioned a second distance along theoptical path 110 wherein the length of the second distance is greaterthan the length of the first distance. The focus minus signal isprovided by focusing the camera assembly on a focal point positioned athird distance along the optical path 110 wherein the length of thethird distance is less than the length of the first distance. The imagesignal, focus plus signal, and focus minus signal are each provided tothe processor 540.

The processor 540 uses the focus plus signal and the focus minus signalto determine the positioning of the slide 1 along the optical path 110necessary for focusing the specimen so that the image signal provided bythe camera 512 will be in focus. More particularly, the processor 540determines whether the received signal is of a magnitude large enough tofocus, whether the image plane lies within the correctable region, andwhich direction to move the slide 1 to focus the image.

Generally, the processor 540 determines the magnitude of the band-passfrequency energy in the focus plus and focus minus signals. Asillustrated in FIG. 10, the image signal will be in focus when theband-pass frequency energy of the focus plus and focus minus signals aresubstantially equal. Accordingly, to determine the proper positioning ofthe slide 1 along the optical path, the processor 540 need onlydetermine how far the slide must be displaced for the energy provided bythe focus plus and focus minus signals to be substantially equal. Itwill be apparent to those skilled in the art that the relativepositioning of the focal point of the camera assembly when providing thefocus plus signal and focus minus signal is determinative of therelationship between their band-pass frequency energy components and thepositioning of the camera assembly for providing a focused image signal.

So that the image signals may be obtained more rapidly, the processor540 is constructed to provide the scan signal to position the motioncontroller 504 in a plurality of X-Y positions to obtain a plurality ofimage signals indicative of a respective plurality of images of aportion of the specimen on the slide 1. The processor 540 may be furtherconstructed to determine the proper positioning of the slide 1 along theoptical path for each of the plurality of image signals obtained. Aftereach of the plurality of image signals has been obtained, the processor540 can determine whether the slide is focused by examining theband-pass frequency component of the focus plus signal and the focusminus signal, as discussed above. If the image signals were not focused,the processor 540 will determine the proper positioning of the slide forfocus and will provide the scan signal to the motion controller 504 tore-position the slide 1 in the X-Y positions of the portions not focusedand, simultaneously, provide the slide focus signal to the motioncontroller 504 to obtain the proper positioning of the slide 1 along theoptical path so that focused image signals are obtained.

A more detailed diagram of the camera assembly 512 is provided in theillustrative diagram of FIG. 11. Therein, an optical transmissionassembly 300 includes an objective lens assembly 302, a first beamsplitter 304 and a second beam splitter 306. The first and second beamsplitters 304 and 306 provide first, second, and third optical paths308, 310, and 312, respectively. The objective lens assembly 302 isconstructed to vary the magnification provided to the specimen on theslide 1. In a presently preferred embodiment of the invention, theobjective lens assembly 302 is responsive to a magnification signalreceived from the processor 540 to select various lenses to vary themagnification. Suitable assemblies for responding to an electric signalto move two or more lenses into and out of position for varying themagnification provided to the specimen may readily be provided by thoseskilled in the art.

A primary camera 314 is positioned to receive a first image of thespecimen on the slide 1 via the first optical path 308. The firstoptical path 308 is the path from point A on the objective 302 to pointB at the CCD of the primary camera 314. The primary camera 314 isresponsive to an activation signal for providing an image signalrepresenting the first image. A focus plus camera 316 is positioned toreceive a second image of the specimen on the slide 1 along a secondoptical path 310. The second optical path 310 is the path from point Aon the objective 302 to point C on the CCD of the focus plus camera 316.The length of the second optical path 310 is less than the length of thefirst optical path 308 by a predetermined length. The focus plus camera316 is also responsive to the activation signal for providing a focusplus signal, wherein the focus plus signal is indicative of the focus ofthe image signal. A focus minus camera 318 is positioned to receive athird image of the object on the slide 1 via a third optical path 312.The third optical path is the path from point A on the objective 302 toa point D on the CCD of the focus minus camera 318. The length of thethird optical path 312 is greater than the length of the first opticalpath 308 by the predetermined length. The focus minus camera 318 isresponsive to the activation signal for providing a focus minus signalthat is also indicative of the focus of the image signal.

As discussed above, the processor 540 determines the band-pass energy ofthe focus plus signal and the focus minus signal to determine the properpositioning of the slide 1 so that the image signals will berepresentative of a focused image of the specimen on the slide.Accordingly, the processor 540 includes first and second identical focusprocessor circuits 400 and 403, as illustrated in FIG. 12. The focusprocessor circuits 400 and 403 each include a band pass filter 404 and406, respectively, for receiving the focus plus and focus minus signals.The band pass filters 404 and 406 are constructed to pass a band-passenergy component of the focus plus and focus minus signals. Eachfiltered signal is multiplied by itself in respective multipliercircuits 408 and 410 so that the resulting signal is always proportionalto the magnitude of the energy. This energy level signal is thenintegrated for each line of active video provided in respectiveintegrators 412 and 414 to provide signals indicative of the totalenergy provided in the band-pass. The output from integrators 412 and414 is sampled by respective sample and hold circuits 416 and 418 beforebeing digitized by an analog-to-digital convertor 420. The processor 540uses the signals from the analog-to-digital convertor 420 to determinethe proper positioning of the slide 1 so that the image signals providedby the primary camera 314 will be representative of a focused image.

In operation, the processor 540 receives an array of focus plus scoresFP(0), FP(1), . . . FP(255), and array of focus minus scores FM(0),FM(1), . . . FM(225), each including 256 elements, one for each line ofa particular field of the camera 512. The focus plus and focus minusarrays provide video signals to the focus processor which are used tocalculate the focus score. Only the first field of the interlaced videoimage is used to calculate the focus score so that the acceptability ofthe image may be determined while the second field is still beingreceived from the camera. In this manner, the focus score is availablebefore the entire image is received. Each line of the image is processedthrough bandpass filters and the energy is integrated and measured bythe analog-to-digital converters.

In order to further understand the filter selection process of theinvention, refer to FIG. 13 where a schematic view of a typical cell isshown. A cell 900 comprises cell cytoplasm 902 and a cell nucleus 904.Typical cell nuclear sizes for pap smears range in area from about 40micrometers squared to 220 micrometers squared. Typical nucleardiameters range from about 4 micrometers to 10 micrometers. In oneexample embodiment of the invention where the magnification of interestis 20×, pixel size is 0.55 micrometers per pixel.

Now referring to FIG. 14, a process for converting physical cell sizeinto electrical band width is schematically illustrated. The conversionfrom physical size into electrical band width may be accomplished byusing the known pixel clock rate from the camera. In this example, thepixel clock rate is 14.1875×10⁶ pixels per second. From the pixel clockrate, the physical size of a cell nucleus may be translated into a timevarying voltage when the camera images the cell nucleus. This techniqueis well known in the art. The pixel time in one example of the inventionis about 70.5×10⁻⁹ seconds. The target for the focus system is between 7and 19 pixels in size. Because some spreading of the object size occursdue to defocused images being used as the stimulus to the cameras formeasuring focus, the size range is increased slightly. The focus systemmay advantageously be made sensitive to objects having a size of from 7to 22 pixels. A nucleus sectioned by a video camera scan line 906 has atime varying modulation 908 in the electrical domain, which correlatesto its size in the spatial domain. The relationship between the spatialdomain and electrical domain is illustrated in FIG. 14 which shows thecell 900 having its nucleus 904 scanned by video lines 906. The scannedcell is then translated into electrical voltages as indicated by plot910 which plots a modulated voltage signal 908 against time.

Referring now to FIG. 15, a time vary voltage of a dark nucleus isgraphically illustrated. The nucleus 904 is analogous to a pulse orsquare function 912 having an interval,t. In this example, shown forillustrative purposes and not by way of limitation of the invention, theinterval t may range from about 493×10⁻⁹ to 1550×10⁻⁹ seconds. FIG. 16shows an inverted pulse 914 which is inversely related to pulse 911.Fourier transformations for such square functions are well known.

Referring now jointly to FIGS. 17 and 18, a Fourier transformation for asquare function is illustrated as employed in one aspect of theinvention. Where a is the smallest nucleus and b is the biggest nucleus,the focus transformation of such pulses then represent spectral energyof objects of the particular size of interest. Using the Fourierrepresentation of these objects, a spectral filter may be chosen whichis sensitive to objects in this size range.

Referring now to FIG. 19, filter response sensitive to objects ofinterest, such as cell nuclei, is schematically illustrated. Filterresponse C may be selected so that the focus system is sensitive to cellnuclei in the size range of interest. Once having the benefit of knowingthe filter response desired for objects in the range of interest astaught by the present invention, a band pass filter may then be designedusing conventional techniques.

Next, a filter operation is performed on each of the four arrays FP, FM,FPnoise, and FMnoise in order to reduce sensitivity to objects that aresmaller than the desired cells that are to be focused on. The filteroperation is sensitive to the vertical size of objects, whereas the bandpass filter on the video lines are sensitive to the horizontal size ofobjects. Accordingly, the system is sensitive to the two dimensionalsize of objects in the focus system. This provides an improved focus andimproves signal-to-noise ratio.

The filter operation can be expressed as follows:

    ______________________________________                                        [FP(0) . . . FP(255)]                                                                             * [Ffk(0) . . . Ffk(4)]                                   [XFPS(2) . . . XFPS(253)]                                                     [FM(0) . . . FM(255)]                                                                             * [Ffk(0) . . . Ffk(4)]                                   [XFMS(2) . . . XFMS(253)]                                                     ______________________________________                                    

The focus plus and focus minus array are each convolved with a filterarray Ffk to correlate the energies of adjacent lines. The filter arrayFfk is selected to provide a low pass filter that looks for objects atleast five lines in size. The filter array Ffk is selected to provide afinite impulse response, low pass filtering of the focus plus and focusminus arrays. The filter kernel is designed to be sensitive to the sizeand type of object that the processor 540 is attempting to detect.Further, the finite impulse response filtering is performed in a mannerso that the resulting filter array eliminates the first and last fewelements of the respective focus plus and focus minus array to eliminateedge effects from the filter.

After filtering the focus plus and focus minus arrays, filtered focusplus and focus minus arrays, XFPS and XFMS, respectively, are createdwith each array including 252 elements. The filtered focus scores arefurther combined with a noise array to eliminate noise that may beprovided by the camera system 512. More particularly, the camera system512 may include noise that results from camera noise, integratorleakage, dust or streaks on the focus camera, or in one of the opticalimage planes. To eliminate this noise, a noise array is generated andcombined with the filtered focus scores. The noise array is generated byfocusing the camera 512 upon a white field, i.e., one with no slide 1 sothat the focus plus and focus minus camera can measure the fixed noisefloor energy within the focus filter band pass. The blank image isviewed in order to obtain a measure of the fixed noise patterns thatwill stimulate the focus processor. The noise arrays of raw focus scoresobtained from viewing the blank image are represented as: [FPnoise(0) .. . FPnoise(255)] for the focus plus array; and, [FMnoise(0) . . .FMnoise(255)] for the focus minus array. The noise floor integration isrelatively consistent and can be measured and subtracted from the energymeasurements made for the individual line scores. This significantlyimproves the signal to noise ratio for each line.

In this regard, a noise plus and noise minus array is measured for thefocus plus and focus minus cameras 316, 318 in the same manner as thefocus plus and focus minus signals, discussed above. The noise plus andnoise minus arrays include an element for each line of the focus plusand focus minus arrays, respectively. The noise plus and noise minusarrays are convolved with the filter array Ffk, as discussed above withthe focus plus and focus minus arrays, to provide filtered noise plusand filtered noise minus arrays, FPNX and FMNX, respectively. Theresulting arrays are filtered noise plus and filtered noise minusarrays, having a one-to-one correspondence with the focus plus and focusminus arrays, respectively. The filter operation on the noise arrays areexpressed as follows:

    ______________________________________                                        [FPnoise(0) . . . FPnoise(255)]                                                                    * [Ffk(0) . . . Ffk(4)]                                  [FPNX(2) . . . FPNX(253)]                                                     [FMnoise(0) . . . FMnoise(255)]                                                                    * [Ffk(0) . . . Ffk(4)]                                  [FMNX(2) . . . FMNX(253)]                                                     ______________________________________                                    

The filter operations are a convolution, shown in the above equations bythe asterisk symbol. The 2 elements on each end of the filtered arraysare excluded since the convolution operation is not defined for theelements on each end of the array. The filtered noise plus and noiseminus arrays, FPNX and FMNX are correspondingly subtracted from thefiltered focus plus and focus minus arrays, XFPS and XFMS, to providerespective focus plus and focus minus signal arrays, FPS and FMS. Thisimproves the S/N ratio. The noise value can be as much as 10%-50% of thetotal signal. Since the noise is static and repeatable, it can beremoved with this method. The noise reduced arrays are as follows:

    [XFPS(2) . . . XFPS(253)]-[FPNX(2) . . . FPNX(253)]=FPS [(2) . . . (253)]

    [XFMS(2) . . . XFMS(253)]-[FMNX(2) . . . FMNX(253)]=FMS [(2) . . . (253)]

The individual elements of the focus plus signal and the focus minussignal arrays are now combined to provide an array of focus scores FS.Now, lines 2 through 253 have scores which are noise reduced and relatedto the two dimensional characteristics of above and below focus images.Each line from the above and below cameras represents a measure (in 2D)of the image frequency content. An array of focus scores can now becalculated as follows: ##EQU1## This step produces a normalized focusscore for each line of the camera 512, except the first and last fewlines that were excluded because of edge filter effects, as discussedabove. Normalization of the focus scores helps to make the dataindependent, i.e., tends to make each score comparable to one anotherregardless of the amount of data used to produce the score. Thisoperation normalizes the focus scores to values somewhere between -1 and+1, to create a spatially distributed set of focus scores.

After the focus plus signal array and focus minus signal array have beencombined as discussed above to produce an array of focus scores, thearray of focus scores is screened to eliminate those scores for whichinsufficient data existed to achieve a meaningful score. This is done byeliminating each score FS(x) for which FPS(x) plus FMS(x) is outside therange of a predetermined threshold. The threshold range is selectedempirically by the lowest signal content image of interest. In apreferred embodiment of the invention, the range is selected to bebetween 3 and 240. Those skilled in the art will appreciate, however,that this range is only illustrative and that any range, including thefull range, may be selected. In one embodiment, favorable results may beobtained using between 1% and 95% of the range. The FS values thatqualify are then averaged to yield a single focus score evaluation forthe image. This single focus score is a number between -1 and +1 whichhas a one-to-one correspondence with the distance necessary to move toput the image into best average focus.

In one aspect of the invention, a focus quality score, FQS(x), may beprovided. The focus quality score comprises the average of FPS(x) plusFMS(x). The focus quality score indicates the signal level of the imageand thereby provides a confidence level for the focus score. If thefocus quality score is below a predetermined level, the focus score isnot accepted as a reliable focus indicator.

After the focus score has been obtained a look up table is consulted fordetermining the distance and direction of movement along the opticalpath necessary to bring the object into focus. As noted above, aparticularly novel aspect of the subject invention is the ability of theprocessor 540 to not only determine whether an image is in focus or outof focus, and not only determine the direction necessary to move thespecimen to bring the image into focus, but to also determine thedistance of motion necessary to bring the specimen into focus. Bydetermining the exact displacement, and direction of displacement,necessary to bring the specimen into focus, the processor 540 maycontrol the motion controller 504 to rapidly return to the position ofany out of focus specimen and may provide the appropriate scan signal sothat the motion controller will position the specimen to be in focus.

To determine the amount of displacement, a look up table comprisingpredetermined correction factors for a given set of optics is employedprior to obtaining any image signals. The correction factors may bederived empirically, for a each set of optics, using known methods. Thecorrection factors in the look up table represent the distance necessaryto move an object into focus. Since the focus scores relate to distance,the correction factors may be related to focus scores. When deriving thecorrection factors, a test image is employed and placed on the motioncontroller. In a presently preferred embodiment of the invention, acalibration to determine the displacement and direction correlation tofocus scores is performed only once when the system is designed andremains the same so long as the component parts of the system are notdisturbed. However, those skilled in the art will appreciate that thecalibration to obtain data correlating the focus scores to the amountand direction of displacement may be performed at any time prior toobtaining image signals.

Using the above-described apparatus, focused image signals may beobtained in a very rapid manner. In a presently preferred embodiment ofthe invention, the motion controller 504 positions the slide 1 at aplurality of predetermined positions for obtaining image signals. Aftereach image signal is obtained, the motion controller 504 immediatelymoves to obtain the next image signal. While the motion controller 504is positioning the slide 1 to obtain the next image signal, theprocessor 540 determines whether the last obtained image signal was infocus. Accordingly, there is a 60 millisecond delay between the timethat the image is taken and the time the image it is read out of theprocessor 540. If the last obtained image was in focus, processor 540identifies the image signal as a focused image signal for use by theremainder of the system. However, if the image signal was not in focus,the processor 540 determines the displacement and direction necessaryfor focus of the specimen. Thereafter, the processor 540 instructs themotion controller 504 to return to the out of focus image and providesthe necessary displacement information so that, when next obtained, theimage will be in focus.

The invention has been described herein in considerable detail in orderto comply with the Patent Statutes and to provide those skilled in theart with the information needed to apply the novel principles and toconstruct and use such specialized components as are required. However,it is to be understood that the invention can be carried out byspecifically different equipment and devices, and that variousmodifications, both as to the equipment details and operatingprocedures, can be accomplished without departing from the scope of theinvention itself.

What is claimed is:
 1. An automated method for checking stageperformance repeatability in an automated optical system having anautomated microscope positioned to view an object on a stage along anoptical path, where the optical path includes an optical axistherethrough, the automated method comprising the steps of:(a) selectinga lens having a first magnification characteristic; (b) inserting acalibration plate into the optical path, where the calibration plateincludes a fiducial having a fiducial center, wherein the fiducial islocated and centered in a field of view byi) grossly positioning thestage in a vicinity of the fiducial, ii) performing a focus pan, iii)determining a location of the fiducial center with respect to theoptical axis, iv) translating the stage in an X, Y plane so as to centerthe fiducial at 0,0 in the field of view, where the X, Y plane isdefined by a top surface of the stage; v) recording the location of thefiducial center; and (c) incrementally moving the stage in a starpattern so as to repeatedly move the fiducial out of the optical axisand then back into line with the optical axis for a predetermined numberof repetitions, where each time the stage returns the fiducial to anoriginal position, an image of the fiducial is captured and processed soas to determine a lateral position in X and Y of the fiducial centerwith respect to the optical axis.
 2. The automated method for checkingstage performance repeatability as set forth in claim 1, where the stepof incrementally moving the stage further comprises the steps of movingthe stage a distance to identify a position repeatabilitycharacteristic.
 3. The automated method for checking stage performancerepeatability of claim 2 further comprising the steps of:i) determininga true Z position of the image by generating a data array containing anX, Y and Z coordinate for each iteration; a) processing the data arrayto determine a set comprising a maximum coordinate and minimumcoordinate in each axis; b) calculating an absolute value of adifference taken between each set so as to determine repeatability ofstage movement in each axis.
 4. The automated method for checking stageperformance repeatability of claim 3 further comprising the steps of:a)checking repeatability along an X axis to a limit of repeatability ofless than 15000 nm; b) checking repeatability along a Y axis to a limitof repeatability of less than 15000 nm; and c) checking repeatabilityalong a Z axis to a limit of repeatability of Z cross coupling of lessthan 2000 nm.
 5. An automated method for checking stage performancerepeatability in an automated optical system having an automatedmicroscope positioned to view an object on a stage along an opticalpath, where the optical path includes an optical axis therethrough, andwhere the stage is initially positioned at an origin, the automatedmethod comprising the steps of:a) selecting a lens having a firstmagnification characteristic; b) inserting a calibration plate into theoptical path, where the calibration plate includes a fiducial and thefiducial is located and centered in a field of view; c) moving the stagealong a Z axis, the Z axis being parallel to the optical axis of thelens, in a negative direction away from the lens a first distance; d)returning the stage to the origin; e) analyzing an actual Z position ofan image; f) moving the stage is in a negative direction for a seconddistance and then returning to the origin; g) analyzing the actual Zposition of the image; h) repeating steps b through g again in exceptreversing a direction of movement of the stage so that the stage ismoved toward the lens; i) storing each Z coordinate in a data array; andj) processing the data array to determine a difference between a minimumand maximum Z coordinate where an absolute value of the difference istaken as a repeatability of the stage.
 6. The automated method forchecking stage performance repeatability of claim 5 further comprisingthe step of checking repeatability along the Z axis to a limit ofrepeatability of less than 2000 nm.
 7. An automated method for checkingturret repeatability in an automated optical system having an automatedmicroscope positioned to view along an optical path, the automatedmethod comprising the steps of:a) selecting a lens having a firstmagnification characteristic; b) inserting a calibration plate into theoptical path, where the calibration plate includes a fiducial and thefiducial is located and centered in a field of view; c) incrementallymoving a turret so as to repeatedly move the fiducial out of an opticalaxis and then back into line with the optical axis for a predeterminednumber of repetitions, where each time a stage returns the fiducial toan origin position, a fiducial image is captured and processed so as todetermine a center of the fiducial in both axes relative to the opticalaxis.
 8. The automated method for checking turret repeatability of claim7 wherein turret movement alternates between clockwise and counterclockwise directions.
 9. The automated method of claim 8 furthercomprising the steps of:a) developing an array of X and Y coordinatesfor each iteration; b) determining a maximum and minimum for each axis;and c) calculating an absolute value for a largest difference as ameasure of turret repeatability
 10. The automated method for checkingturret repeatability of claim 9 further comprising the step of checkingrepeatability to a limit of repeatability of less than 7000 nm.
 11. Anautomated method for testing objective centration and parfocality in anautomated microscope system having multiple objective lenses, whereineach objective has its own optical axis, the automated method comprisingthe steps of:a) selecting a lens having a first magnificationcharacteristic; b) inserting a calibration plate into a first opticalpath, where the calibration plate includes a fiducial and the fiducialis located and centered in a field of view, and obtaining a first set ofX,Y and Z coordinates; c) selecting a lens having a second magnificationcharacteristic; d) inserting the calibration plate into a second opticalpath, where the calibration plate includes a fiducial and the fiducialis located and centered in a field of view, and obtaining a second setof X,Y and Z coordinates; and e) subtracting the first set of X,Y and Zcoordinates from the second set of X,Y and Z coordinates, where an X andY difference comprises a centration characteristic of the objectives anda Z difference comprises a parfocality factor.
 12. The automated methodfor checking stage performance repeatability of claim 11 furthercomprising the steps of:a) checking repeatability of centration to alimit of repeatability of less than 25000 nm; b) checking repeatabilityof parfocality to a limit of repeatability of less than 15000 nm.
 13. Anautomated apparatus for checking stage performance repeatability in anautomated optical system having an automated microscope positioned toview an object on a stage along an optical path, where the optical pathincludes an optical axis therethrough, the automated apparatuscomprising:a) a lens having a first magnification characteristic; b) acalibration plate inserted into the optical path, where the calibrationplate includes a fiducial having a fiducial center, wherein the fiducialis located and centered in a field of view; c) means, coupled to thestage, for grossly positioning the stage in a vicinity of the fiducial;d) means, coupled to the automated microscope, for performing a focuspan; e) means, coupled to the stage, for determining a location of acenter of the fiducial with respect to an optical axis; f) means,coupled to the stage, for translating the stage in an X, Y plane so asto center the fiducial in the field of view, where the X, Y plane isdefined by a top surface of the stage; g) means, coupled to the stage,for recording the location of the fiducial center; and h) means, coupledto the stage, for incrementally moving the stage in a star pattern so asto repeatedly move the fiducial out of the optical axis and then backinto line with the optical axis for a predetermined number ofrepetitions, where each time the stage returns the fiducial to anoriginal position, an image of the fiducial is captured and processed soas to determine a lateral position of a fiducial center in the X,Y planewith respect to the optical axis.
 14. The automated apparatus forchecking stage performance repeatability as set forth in claim 13, wherethe means for incrementally moving the stage further comprises a meansfor moving the stage a distance suitable to identify a positionrepeatability characteristic.
 15. The automated apparatus for checkingstage performance repeatability of claim 13 further comprising:a) means,coupled to the stage, for determining a true Z position of the image bygenerating a data array containing an X, Y and Z coordinate for eachiteration; b) means, coupled to the means for determining a true Zposition, for processing the data array to determine a set comprising amaximum coordinate and minimum coordinate in each axis; and c) means,coupled to the means for processing the data arrays, for calculating anabsolute value of a difference taken between each set so as to determinethe repeatability of stage movement in each axis.
 16. The automatedapparatus for checking stage performance repeatability of claim 15further comprising:a) means, coupled to the means for processing thedata arrays, for checking repeatability along an X axis to a limit ofrepeatability of less than 15000 nm; b) means, coupled to the means forprocessing the data arrays, for checking repeatability along a Y axis toa limit of repeatability of less than 15000 nm; and c) means, coupled tothe means for processing the data arrays, for checking repeatabilityalong a Z axis to a limit of repeatability of Z cross coupling of lessthan 2000 nm.
 17. An automated apparatus for checking stage performancerepeatability in an automated optical system having an automatedmicroscope positioned to view an object on a stage along an opticalpath, where the optical path includes an optical axis therethrough, andwhere the stage is initially positioned at an origin, the automatedapparatus comprising:a) a lens having a first magnificationcharacteristic; b) a calibration plate inserted into the optical path,where the calibration plate includes a fiducial and the fiducial islocated and centered in a field of view; c) means, coupled to the stage,for moving the stage along a Z axis, the Z axis being parallel to theoptical axis of the lens, in a negative direction away from the lens afirst distance; d) means, coupled to the stage, for returning the stageto the origin; e) means, coupled to the stage, for analyzing an actual Zposition of an image; f) means, coupled to the stage, for moving thestage in a negative direction for a second distance and then returningto the origin; g) means, coupled to the stage, for analyzing the actualZ position of the image; h) means, coupled to the stage, for reversing adirection of movement of the stage so that the stage is moved toward thelens; i) means for storing each Z coordinate in a data array; and j)means, coupled to the storing means, for processing the data array todetermine a difference between a minimum and maximum Z coordinate wherean absolute value of the difference is taken as a repeatability of thestage.